A crystalline icariogen monohydrate and a preparation method thereof

Abstract

The application belongs to the field of medicine crystal form, and particularly relates to a icariin aglycone hydrate and a preparation method thereof. The icariin aglycone hydrate crystal provided by the application has X-ray diffraction spectrum peaks at 13.80+0.2°, 16.24+0.2°, 21.22+0.2°, 23.09+0.2° and 43.33+0.2°, expressed by 2θ, using Cu-Kα radiation; the icariin aglycone monohydrate crystal provided by the application has excellent properties, high solubility and stability, provides a high-quality raw material selection for the preparation of icariin aglycone, and the preparation method is simple to operate, easy to control and suitable for industrial amplification.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical cocrystals, in particular to icaritin monohydrate crystal and a preparation method thereof. BACKGROUND

[0002] Icaritin (IT), also known as icariin, is a monomeric component of multi-hydroxyl flavones in Epimedium, which is a plant of Berberidaceae. The structural formula is shown in the following formula (I):

[0003]

[0004] Pharmacological studies show that icaritin has stronger anti-osteoporosis activity than other flavone glycosides in Epimedium. Icaritin can promote the activity of osteoblasts and inhibit the activity of osteoclasts in vitro. Traditional Chinese medicine Epimedium has the effects of tonifying kidney and yang, strengthening muscles and bones, dispelling wind and dampness, washing sores and killing insects, and relieving fatigue and pain. Icaritin, as one of the main effective components, has attracted the attention of many scholars at home and abroad in recent years, and its pharmacological effects have been extensively studied. So far, it has been found that the main physiological activity of icaritin is to improve the function of the cardiovascular system, enhance the body's immunity, and regulate endocrine, and it also has the effects of anti-tumor, anti-liver toxicity, anti-hypoxia reoxygenation, and bone strengthening.

[0005] Although icaritin has good clinical application prospects, its poor water solubility greatly limits its clinical application. At present, the research on solving the solubility of icaritin mainly focuses on two aspects: one is to study the preparation of new crystal forms of icaritin or its derivatives, and the other is to improve the solubility through the means of preparation. For example, Jia Dongsheng et al. significantly improved the solubility of raw materials and physical mixtures by preparing icaritin phospholipid complex (Jia Dongsheng, Zhao Jiangli, Shi Feng, et al. Preparation of icaritin phospholipid complex and research on solid dispersion [J]. Chinese Herbal Drugs, 2010, 9(41)), but this method has the problem that the dissolution medium may destroy the structure of the drug or the evaporation of the dissolution medium; Wang Jinyan et al. studied the solubilization of icaritin by microemulsion technology (Jinyan, Chen Yan, Zhang Zhenhai, et al. Preliminary study on the intestinal absorption characteristics of icaritin self-microemulsion in Caco-2 cell model [J]. Chinese Herbal Drugs, 2012, 3(43)), which can improve the solubility and absorption of icaritin, but a large amount of emulsifying agent and co-emulsifying agent are used in the process, which has certain quality and safety risks.

[0006] There are few reports on the study of icariin aglycone crystal form, and patent CN101200743A reports the preparation of icariin aglycone crystal, but does not improve its solubility. Patent CN104860958A reports the crystal form A and crystal form B of dehydroicaritin, and the results show that the solubility of dehydroicaritin is improved compared with that of amorphous powder, but there is no significant improvement. Patent CN103936705A discloses four icaritin solvates and one anhydrous crystal form B, and reports that the solvate crystal forms are unstable and are easily converted into anhydrous crystal form B. Patents CN104230870A and CN104945364A disclose two icaritin hydrate crystal forms, and study the light stability of icaritin hydrate crystal form and anhydrous crystal form B, and report that anhydrous crystal form B is not stable under light, and the hydrate crystal form has improved light stability to a certain extent. Patent CN112294765A discloses an icaritin amorphous form, which has improved solubility and bioavailability compared with icaritin crystal form, but the amorphous form generally has poor stability, which is not conducive to its application in drugs.

[0007] The solubility and stability of the reported icaritin crystal forms have not been well solved, and the improvement of the medicinal effect is not ideal, so it is still necessary to study new crystal forms which can significantly improve the solubility and stability of icariin aglycone, fundamentally solve the solubility and stability problems of icariin aglycone, and minimize the addition of auxiliary materials in the preparation process and minimize the drug hazards. SUMMARY

[0008] In view of the problems existing in the prior art, the present application successfully prepares an icariin aglycone monohydrate crystal by co-crystal technology, which can significantly improve the solubility of icariin aglycone and has good physicochemical stability.

[0009] The specific technical content of the present application is as follows:

[0010] In the first aspect of the present application, an icariin aglycone monohydrate crystal is provided, characterized in that the molar ratio of icariin aglycone to water in the crystal unit structure is 1:1.

[0011] Preferably, the icariin aglycone monohydrate crystal has characteristic peaks at 13.80±0.2°, 16.24±0.2°, 21.22±0.2°, 23.09±0.2° and 43.33±0.2° in the X-ray diffraction spectrum expressed in 2θ using Cu-Kα radiation.

[0012] Preferably, the icariellin monohydrate crystal has an X-ray diffraction spectrum using Cu-Ka radiation with characteristic peaks at 12.28±0.2°, 13.80±0.2°, 16.24±0.2°, 21.22±0.2°, 23.09±0.2°, 23.99±0.2°, 26.28±0.2°, 31.12±0.2°, 33.78±0.2°, 43.33±0.2°, as shown in the X-ray powder diffraction spectrum.

[0013] Preferably, the icariellin monohydrate crystal has an X-ray diffraction spectrum using Cu-Ka radiation with characteristic peaks at 12.28±0.2°, 13.80±0.2°, 16.24±0.2°, 21.22±0.2°, 23.09±0.2°, 23.99±0.2°, 26.28±0.2°, 31.12±0.2°, 33.78±0.2°, 43.33±0.2°, as shown in the X-ray powder diffraction spectrum. Figure 1

[0014] Preferably, the icariellin monohydrate crystal has an X-ray diffraction spectrum using Cu-Ka radiation with characteristic peaks at 12.28±0.2°, 13.80±0.2°, 16.24±0.2°, 21.22±0.2°, 23.09±0.2°, 23.99±0.2°, 26.28±0.2°, 31.12±0.2°, 33.78±0.2°, 43.33±0.2°, as shown in the X-ray powder diffraction spectrum. α = 87.719 (2) °, β = 88.772 (2) °, γ = 69.076 (2) °, cell volume V = 1 1 1 1. 1 (3) A3, Z = 2, Dc = 1. 34 g / cm3.

[0015] In the second aspect of the present application, a preparation method of icariellin monohydrate crystal is provided, and the specific steps are as follows: icariellin is dissolved in a mixed solution of organic solvent and water, heated and stirred, cooled and crystallized, filtered and dried to obtain icariellin monohydrate crystal.

[0016] Preferably, the organic solvent is selected from one or more of acetonitrile, tetrahydrofuran, methanol, ethanol; further preferably, the organic solvent is selected from one of acetonitrile and methanol.

[0017] Preferably, the mass-volume ratio of icariellin to water is 71-135:1, mg / mL; preferably 90-106:1, mg / mL.

[0018] Preferably, the volume ratio of the organic solvent to water is 3-7:1, preferably 4:1.

[0019] Preferably, the temperature of the dissolution and heating is 35-42°C.

[0020] Preferably, the temperature of the cooling and crystallization is 5-20°C.

[0021] Preferably, the crystallization time is 12-30 hours.

[0022] Preferably, the drying temperature is 25-40°C, and the drying time is 11-15 hours.

[0023] ​More preferably, the specific steps of the preparation method of the icariin monohydrate crystal are as follows: dissolve icariin in a mixed solution of organic solvent / water, heat and stir to dissolve at 35-42°C, cool naturally to 5-20°C for crystallization for 12-30 hours, filter, wash the filter cake, and dry to obtain icariin monohydrate crystal.

[0024] Preferably, the solvent for washing the filter cake is selected from ethanol and tetrahydrofuran.

[0025] In a third aspect, the present invention provides a pharmaceutical composition comprising the icariin monohydrate crystals described herein and other pharmaceutically acceptable components.

[0026] Preferably, the pharmaceutical composition is prepared by means of: using standard and conventional techniques, combining icariin monohydrate crystals with a pharmaceutically acceptable solid or liquid carrier, and arbitrarily combining them with pharmaceutically acceptable excipients and formulations to prepare a pharmaceutical dosage form.

[0027] Preferably, the other pharmaceutically acceptable components include pharmaceutically active ingredients, excipients, fillers, etc., that can be used in combination.

[0028] Preferably, the dosage form of the pharmaceutical composition includes, but is not limited to, sprays, tablets, capsules, powder for injection, and injections.

[0029] In a fourth aspect, the present invention provides the use of icariin monohydrate crystals as an active ingredient in the preparation of drugs for treating liver injury, liver fibrosis, tumors, etc.

[0030] Confirmation of crystal structure:

[0031] X-ray crystal data were collected on a Rigaku XtaLAB Synergy instrument in Japan at a test temperature of 293(2) K. CuKa radiation was used, and data were collected via ω-scan and Lp correction was performed. The crystal structure was calculated using the ShelXT program in the 01ex2 software. The structure parameters and atom types were corrected using the least squares method in the ShelXL program. The positions of all hydrogen atoms were obtained using geometric calculation and the Fourier difference method. The goodness of fit (goof value) was 1.019, close to 1.0, indicating that the weighting scheme was appropriate and the structure was accurate. The crystallographic data of the icariin monohydrate prepared in this invention are shown in Table 1. The ORTEP diagram and crystal packing diagram of the icariin monohydrate of this invention are attached. Figure 2 Appendix Figure 3 As shown.

[0032] Table 1. Main crystallographic data of icariin monohydrate

[0033]

[0034] The X-ray powder diffraction testing instrument and testing conditions in the present application are as follows: X-ray powder diffraction instrument: PANalytical EMPYREAN; Cu-Ka; sample table: flat plate; incident light path: BBHD; diffraction light path: PLXCEL; voltage: 45kv, current: 40mA; divergence slit: 1 / 4; anti-scattering slit: 1; soller slit: 0.04 rad; step length: 0.5s; scanning range: 3-50°. According to the crystallographic data, the characteristic peaks in the corresponding X-ray powder diffraction pattern (Cu-Ka) of icariellin monohydrate are shown in Table 2 and Table 2. Figure 1

[0035] Table 2. Main PXRD peaks of icariellin monohydrate

[0036]

[0037]

[0038] The samples prepared in Examples 1-5 all meet the X-ray powder diffraction spectrum.

[0039] The beneficial effects of the present application are as follows:

[0040] The method for preparing icariellin monohydrate co-crystals provided by the present application is simple to operate, and the prepared crystals have high purity. The icariellin monohydrate crystals provided by the present application have good stability, and significantly improve the solubility of icariellin, thereby providing a high-quality raw material selection for the preparation of icariellin. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 X-ray powder diffraction pattern of icariellin monohydrate.

[0042] Figure 2 ORTEP diagram of icariellin monohydrate.

[0043] Figure 3 Stacked diagram of icariellin monohydrate. DETAILED DESCRIPTION

[0044] The present application is further illustrated by the following examples, and it should be understood that the examples of the present application are only used to illustrate the present application, and are not a limitation of the present application, so that simple improvements of the present application under the premise of the method of the present application are within the scope of the present application.

[0045] The raw material icariellin in the present application can be prepared according to any method in the prior art or purchased from commercially available products (purity ≥98%), and the remaining reagent materials are commercially available. ​

[0046] I. Preparation of the crystalline form

[0047] Example 1

[0048] Dissolve 368.4 mg of icariogenin in a mixed solution of 14.8 mL of acetonitrile and 3.7 mL of water, stir at 40°C until completely dissolved, slowly cool to 10-15°C and stand for 16-18 hours to crystallize, filter, wash the filter cake with ethanol, and vacuum dry at 30°C for 12-13 hours to obtain icariogenin monohydrate crystals, yield: 98.6%, purity: 99.99%.

[0049] Example 2 (90,4)

[0050] Dissolve 368.4 mg of icariogenin in a mixed solution of 16.4 mL of acetonitrile and 4.1 mL of water, stir at 40-42°C until completely dissolved, slowly cool to 15-20°C and stand for 1 day to crystallize, filter, wash the filter cake with tetrahydrofuran, and vacuum dry at 25°C for 14-15 hours to obtain icariogenin monohydrate crystals, yield: 98.2%, purity: 99.98%.

[0051] Example 3 (106,4)

[0052] Dissolve 368.4 mg of icariogenin in a mixed solution of 13.9 mL of methanol and 3.5 mL of water, stir at 35°C until completely dissolved, slowly cool to 5-10°C and stand for 12-13 hours to crystallize, filter, wash the filter cake with ethanol, and vacuum dry at 40°C for 11-12 hours to obtain icariogenin monohydrate crystals, yield: 97.9%, purity: 99.96%.

[0053] Example 4 (71,3)

[0054] Dissolve 368.4 mg of icariogenin in a mixed solution of 15.6 mL of tetrahydrofuran and 5.2 mL of water, stir at 38-40°C until completely dissolved, slowly cool to 10-15°C and stand for 16-18 hours to crystallize, filter, wash the filter cake with tetrahydrofuran, and vacuum dry at 30°C for 12-13 hours to obtain icariogenin monohydrate crystals, yield: 96.4%, purity: 99.96%.

[0055] Example 5 (135,7)

[0056] Dissolve 368.5 mg of icariogenin in a mixed solution of 19.0 mL of ethanol and 2.7 mL of water, stir at 40°C until completely dissolved, slowly cool to 10-15°C and stand for 30 hours to crystallize, filter, wash the filter cake with ethanol, and vacuum dry at 30°C for 12-13 hours to obtain icariogenin monohydrate crystals, yield: 97.2%, purity: 99.97%.

[0057] Comparative Example 1 (D1 - single crystal)

[0058] 368.4 mg of icariogenin was dissolved in 30 mL of acetone, filtered, about 15 mL of distilled water was added to the filtrate, dissolved at 75°C, and crystallized at 20°C. After crystallization for 24 hours, light yellow crystals were obtained by filtration. The obtained crystals were continuously dried at 80°C until the weight of the crystals no longer changed, to obtain icariogenin crystals, with a yield of 91.7% and a purity of 99.87%.

[0059] Comparative Example 2 (D6 - hemihydrate)

[0060] 1 g of icariogenin crude product and 70 mL of acetone were placed in a 100 mL beaker, dissolved by stirring in a warm water bath, and stirred at 40°C until completely dissolved. Then the solution was quickly added to a beaker containing 400 mL of purified water at room temperature, and stirred vigorously for 10 min, and a large amount of solid was precipitated. After the solution was cooled to room temperature, it was filtered, and the filter cake was dried in a 25°C air-drying oven for 48 hours to obtain icariogenin hemihydrate, with a yield of 89.8% and a purity of 99.86%.

[0061] Comparative Example 3 (D6 - monohydrate)

[0062] 1 g of icariogenin crude product and 70 mL of acetone were placed in a 1 L flask, dissolved by stirring in a warm water bath, and stirred at 40°C until completely dissolved. Then 400 mL of purified water was quickly added under stirring, and stirred for 10 min, and a large amount of solid was precipitated. After the solution was cooled to room temperature, it was filtered, and the filter cake was dried in a 25°C air-drying oven for 48 hours to obtain icariogenin monohydrate, with a yield of 92.5% and a purity of 99.82%.

[0063] II. Verification Examples

[0064] 1. Stability test

[0065] The stability test method of the present application was carried out according to the guidance method for stability investigation in the fourth part of the Chinese Pharmacopoeia (2020 edition). The high temperature test condition was 60°C, the strong light irradiation test condition was 4500lx±500lx, and the high humidity test condition was 92.5%. The purity was detected by HPLC method, and 3 parallel experiments were carried out, and the average value was taken. The specific detection results are shown in Table 3.

[0066] Table 3 Stability test results of icariogenin monohydrate under light, high temperature and high humidity conditions

[0067]

[0068] At the same time, it was found that Examples 1-5 of the present application had similar stability test results.

[0069] 2. Solubility experiment

[0070] Method: 10 ml of medium (water, 0.01 mol / L HC1 solution) was respectively taken in a Schlenk flask, an excess of the sample to be tested was added, the Schlenk flask was sealed and placed in a 25℃ constant temperature water bath for stirring for 1 hour, filtered through a filter membrane, and the filtrate was taken; the absorbance was measured at a wavelength of 270 nm, and the solubility was calculated by testing the absorbance of the standard control.

[0071] Table 4 Solubility of icariogenin methanol solvate in different media (μg / mL)

[0072]

[0073]

[0074] At the same time, it was found that the examples 1-5 had similar solubility test results.

[0075] III. Biological activity experiment

[0076] 1. Materials

[0077] 1.1 Drug

[0078] Example 1 and comparative examples 1-3 are self-made crystal forms.

[0079] 1.2 Animals

[0080] 30 Beagle experimental dogs, half male and half female, weighing (12-15 kg), were provided by Guangzhou Medical Research Institute Co., Ltd.

[0081] 2. In vivo pharmacokinetics experiment of Beagle experimental dogs

[0082] After 30 Beagle dogs were adaptively fed for two weeks and quarantined, 24 healthy normal ones were selected and divided into four groups, which were respectively given the crystal forms of example 1 and comparative examples 1-3, and orally administered at a dose of 15 mg / kg. Blood was collected from the peripheral vein at 0, 0.5, 1, 2, 4, 6, 8 and 12 hours, and plasma was obtained by centrifugation at 2-8℃ for 10 minutes. The concentration of icariin in the plasma of Beagle dogs was determined by hydrolysis of β-glucuronidase and LC-MS / MS method (reference: Huang Y, Liu YJ, Hu L, et al. Determination of the concentration of acoradin in Beagle plasma by LC-MS / MS method[C]. The Seventh National Toxicology Conference and the Eighth Hubei Science and Technology Forum. 2015.). The measured pharmacokinetic parameters are shown in Table 5, wherein the area under the blood concentration-time curve (AUC) was calculated by trapezoidal method, and the elimination half-life (t 1 / 2) Peak concentration (C max ) and time to peak (T max ) were calculated using Best Fit.

[0083] Table 5 Pharmacokinetic parameters of beagle dogs after oral administration n = 6

[0084]

[0085] Note: Group 1 was administered Example 1; Group 2 was administered Comparative Example 1; Group 3 was administered Comparative Example 2; Group 4 was administered Comparative Example 3.

Claims

1. A crystalline form of icariin monohydrate, characterized in that, Using Cu-Kα radiation, the X-ray diffraction pattern, expressed as 2θ, shows characteristic peaks at 13.80±0.2°, 16.24±0.2°, 21.22±0.2°, 23.09±0.2°, and 43.33±0.2°. The molar ratio of icariin to water in the crystal unit structure is 1:

1.

2. The icariin monohydrate crystal according to claim 1, characterized in that, Using Cu-Kα radiation, its main crystallographic parameters are: triclinic crystal system, space group P-1; unit cell parameters are: a =9.3820(2)Å, b =12.1778(2)Å, c =17.5462(4)Å, α=87.719(2)°, β=88.772(2)°, γ=69.076(2)°, cell volume V =1870.95(7)Å 3 .

3. The icariin monohydrate crystal according to claim 1, characterized in that, Using Cu-Kα radiation, the X-ray diffraction pattern, expressed as 2θ, shows characteristic peaks at 12.28±0.2°, 13.80±0.2°, 16.24±0.2°, 21.22±0.2°, 23.09±0.2°, 23.99±0.2°, 26.28±0.2°, 31.12±0.2°, 33.78±0.2°, and 43.33±0.2°.

4. The icariin monohydrate crystal according to claim 1, characterized in that, Its characteristic peaks have the X-ray powder diffraction pattern shown in Figure 1.

5. A method for preparing icariin monohydrate crystals according to any one of claims 1-4, characterized in that, The specific steps are as follows: Icariin is dissolved in a mixed solution of organic solvent and water, heated and stirred, cooled to crystallize, filtered and dried to obtain icariin monohydrate crystals, wherein the organic solvent is selected from one or more of acetonitrile, tetrahydrofuran, methanol and ethanol.

6. The preparation method according to claim 5, characterized in that, The mass-to-volume ratio of icariin to water is 71–135:1, mg / mL; the volume ratio of the organic solvent to water is 3–7:

1.

7. The method according to claim 5, characterized in that, The melting and heating temperature is 35–42°C.

8. The preparation method according to claim 5, characterized in that, The specific steps are as follows: Dissolve icariin in a mixed solution of organic solvent / water, heat and stir at 35-42℃ to dissolve, cool naturally to 5-20℃ for 12-30 hours to crystallize, filter, wash the filter cake, and dry to obtain icariin monohydrate crystals.

Images